The tumor suppressor pathway involving the P53 transcriptional regulator is inactivated in almost all human cancers. In many cancers this is due to attenuation of wild type P53 function by aberrant expression of its negative regulators Hdm2 and HdmX. Mouse models and in vitro transfection studies show that the control of P53 stability and functional output are very sensitive to small changes in the concentrations of its proposed regulatory factors. Therefore, precise quantification of the intracellular concentrations of P53 and its negative regulators including H/Mdm2, H/Mdmx and the deubiquitylating enzyme HAUSP are critical for developing accurate models of P53 regulation. The four Specific Aims of this grant employ quantitative biochemical strategies, mouse molecular genetics, and siRNA functional analyses and screens to elucidate the molecular mechanisms that regulate the P53 tumor suppressor pathway in normal and cancer cells prior to and following stress. Specific Aims 1 and 2 quantify P53 and its regulators in the cytoplasm, nucleus, and on chromatin. The studies are designed to test a new model for P53 regulation in which P53-mediated activation of the H/Mdm2 ubiquitin ligase acts in a positive feedback loop to eliminate H/Mdmx, the primary P53 transcriptional antagonist. Specific Aim 3 uses mouse models to test the positive feedback loop hypothesis, and to analyze structure-function relationships in Mdm2 and MdmX. Specific Aim 4 proposes to examine the mechanisms by which activated oncogenes can inactivate the P53 pathway in the substantial fraction of tumors that express wild type P53. It also utilizes an siRNA screen to identify new regulators that mediate proteasomal degradation of P53, Hdm2 and HdmX. Cancer clearly results from activating mutations in oncogenes that accelerate growth or increase survival, and inactivating mutations that disable tumor suppressors such as P53. However, we now understand that the P53 gene is normal in almost 50% of cancers, and that oncogenic mutations disable P53 function. Thus, activating P53 in such cancers could provide a huge opportunity to improve cancer treatment for a substantial number of cancer patients. Achieving this goal requires understanding how P53 is regulated in normal cells to deduce how it is inactivated in tumors. Only then can we develop therapies to target the genetic defects that disable P53 in a particular tumor. This proposal is designed to provide such information.